Cell differentiation is the central characteristic of tissue morphogenesis which initiates during embryogenesis, and continues to various degrees throughout the life of an organism in adult tissue repair and regeneration mechanisms. The degree of morphogenesis in adult tissue varies among different tissues and is related, among other things, to the degree of cell turnover in a given tissue.
The cellular and molecular events which govern the stimulus for differentiation of cells is an area of intensive research. In the medical and veterinary fields, it is anticipated that the discovery of the factor or factors which control cell differentiation and tissue morphogenesis will advance significantly medicine's ability to repair and regenerate diseased or damaged mammalian tissues and organs. Particularly useful areas for human and veterinary therapeutics include reconstructive surgery and in the treatment of tissue degenerative diseases including arthritis, emphysema, osteoporosis, cardiomyopathy, cirrhosis, degenerative nerve diseases, inflammatory diseases, and cancer, and in the regeneration of tissues, organs and limbs. In non-mammalian systems, such as insect systems, the discovery of such factors provide the basis for developing potent insecticides by, for example, identifying compounds which inhibit or otherwise interfere with the morphogenetic effect of these factors in the insect. (In this and related applications, the terms "morphogenetic" and "morphogenic" are used interchangeably.)
A number of different factors have been isolated in recent years which appear to play a role in cell differentiation. Recently, various members of the structurally related proteins of the transforming growth factor-.beta. (TGF-.beta.) superfamily of proteins have been identified as true tissue morphogens.
This "family" of proteins, constituting a distinct subfamily within the TGF-.beta. superfamily of structurally related proteins, share substantial amino acid sequence homology within their morphogenically active C-terminal domains, including a conserved six or seven cysteine skeleton, and are capable of inducing tissue-specific morphogenesis in a variety of organs and tissues. The proteins apparently contact and interact with progenitor cells e.g., by binding suitable cell surface molecules, predisposing or otherwise stimulating the cells to proliferate and differentiate in a morphogenically permissive environment. The morphogens are capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new organ-specific tissue, including any vascularization, connective tissue formation, and nerve innervation as required by the naturally occurring tissue.
A number of proteins useful in tissue morphogenesis have been identified to date, including proteins originally identified as bone inductive proteins, such as the OP-1, (also referred to in related applications as "OP1"), OP-2 (also referred to in related applications as "OP2"), OP-3 and the CBMP2 proteins, as well as amino acid sequence-related proteins such as BMP5, BMP6 and its murine homolog, Vgr-1, dpp and 60A (from Drosophila), Vgl (from Xenopus), and GDF-1 (from mouse.) See, for example, PCT documents US92/01968 and US92/07358 (published as WO 92/15323 and WO 93/04692, respectively), the disclosures of which are incorporated herein by reference. These TGF-.beta. superfamily members comprise a distinct subfamily of proteins different from other members of the TGF-.beta. superfamily in that the family of morphogenic proteins are able to induce the full cascade of events that result in formation of functional tissue rather than merely inducing formation of fibrotic (scar) tissue as, for example, TGF-.beta. does in many cases. Specifically, members of the morphogen family of proteins are capable of all of the following in a morphogenically permissive environment: stimulating cell proliferation and cell differentiation, and supporting the growth and maintenance of differentiated cells. The morphogenic proteins apparently may act as endocrine, paracrine or autocrine factors.
These proteins are capable of significant species "crosstalk." For example, dpp and 60A, two Drosophila proteins, can induce endochondral bone formation at a non-bony site in a standard rat bone formation assay. In their native form, however, the proteins appear to be tissue-specific, each protein expressed in or provided to one or only a few tissues or, alternatively, expressed only at particular times during development. For example, OP-1 is expressed and/or present primarily in tissues of urogenital origin or bone tissue, although it has been identified in mammary, salivary gland tissue, and reproductive tissues, as well as in gastrointestinal tract tissue. GDF-1 appears to be expressed primarily in neural tissue, while OP-2 appears to be expressed in early (e.g., 8-day) mouse embryo. The endogenous morphogens may be synthesized by the cells on which they act, by neighboring cells, or by cells of a distant tissue, the secreted protein being transported to the cells to be acted on.
Recently, the genetic sequences encoding receptors to several members of the TGF-.beta. protein superfamily have been described. Lin et al. (1992) Cell 68:775-785, disclose the expression cloning of the TGF-.beta. Type II receptor. Several groups have described genetic sequences encoding various activin (Type II) receptors in different species, including mouse, rat, xenopus, and human. The overall amino acid sequence homology between these activin receptors is 50-80%. See, Matthews et al. (1991) Cell 65:973-982 and international patent application WO 92/20793, published Nov. 26, 1992, disclosing the "ActR II" sequence; Attisano et al., (1992) Cell 68:97-108, disclosing the "ActR-IIB" sequence; and Legerski et al. (1992) Biochem Biophys. Res.Comm'n 183:672-679. By amino acid sequence homology to the TGF-.beta. and activin Type II receptor sequences, the daf-1 gene, (Georgi et al. (1990) Cell 61:635-645), identified in C. elegans and having no known ligand to date, also is believed to encode a receptor for a TGF-.beta. superfamily protein member. These disclosed receptors for TGF-.beta. and activin are distinct from cell surface receptors capable of specific binding interaction with the morphogens described herein, and do not bind these morphogens significantly (see, for example, Legerskie et al. (1992) Biochem. Biophys.Res. Comm'n. 183:672-679 and Attisano et al., (1992) Cell 68:97-108.)
To date, the molecule or molecules with which the morphogens described herein interact on the cell surface have not yet been identified. Identification of these cell surface molecules, with which the morphogens interact and through which they may mediate their biological effect, is anticipated to significantly enhance elucidation of the molecular mechanism of tissue morphogenesis and to enable development of morphogen receptor binding "analogs", e.g., compounds (which may or may not be amino acid-based macromolecules) capable of mimicking the binding affinity of a morphogen for its receptor sufficiently to act either as a receptor binding agonist or antagonist. These "analogs" have particular utility in therapeutic, diagnostic and experimental research applications.
It is an object of this invention to provide nucleic acid and amino acid sequences encoding a morphogen binding cell surface receptor, including allelic, species, chimeric and mutant variants thereof. Another object is to provide methods for identifying genes in a variety of species and/or tissues, and in a variety of nucleic acid libraries encoding morphogen receptors which share little or no substantial amino acid identity with the extracellular domains of known TGF-.beta. or activin receptor molecules. Still another object is to provide means for the expression of morphogen receptors, including truncated forms thereof, using recombinant DNA technology. Yet another object is to provide means for designing biosynthetic morphogen receptor-binding ligands and/or for identifying natural-occurring ligands, including morphogen agonists and antagonists, using the morphogen receptor molecules of this invention, and analogs thereof. Still another object is to provides means and compositions for modulating the endogenous expression or concentration of these receptor molecules. Yet another object is to provide compositions and methods for creating useful insecticides. These and other objects and features of the invention will be apparent from the description, drawings and claims which follow.